Lab Session 9

1. Lab Session 9 IUG, 2012 TMZ

2. SDS-PAGE Part I

3. SDS-PAGE (PolyAcrylamide Gel Electrophoresis) • SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology: • to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). • to separate proteins according to their size.

4. Electrophoretic Theory In an electric field: • Charged molecules behave in a predictable manner. + Positively charged molecules will move towards the negative pole + Negatively charged molecules move towards the positive pole.

5. ..SDS • SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein. • The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein.

7. Steps in SDS-PAGE • Extract Protein • Solubilize and Denature Protein • Separate Proteins on a gel • Stain proteins (visualization) • Analyze and interpret results

9. PAGE • If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size. • However, if the proteins are put into an environment that will allow different sized proteins to move at different rates. • The environment is polyacrylamide. • The entire process is called polyacrylamide gel electrophoresis (PAGE).

10. …In a polyacrylamide gel • Small molecules move through the polyacrylamide forest faster than big molecules

11. Why using Polyacrylamide? • It is inert • Can easily be made up at a different concentrations to produce different pore sizes.

12. …SDS-PAGE • The end result of SDS-PAGE has two important features: • all proteins contain only primary structure & • all proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.

13. The actual bands are equal in size, but the proteins within each band are of different sizes.

14. Sample of SDS- PAGE

15.  Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.

16. Polyacrylamide Gel Cathode Anode Proteins separated by molecular weight

17. The gel matrix • Discontinuous buffer system? • Laemmli buffer system: • Buffer in gel & tank are different • Astacking gel at pH 6.8, buffered by Tris-HCl • A running gel buffered to pH 8.8 by Tris-HCl and an electrode buffer at pH 8.3 • Note: Stacking gel has a low conc. of polyacrylamide, while the running gel has a high conc., of polyacrylamide to slow down the movement of proteins

18. The role of Glycine.. • Glycine can exist in three different charge states, positive, neutral or negative depending on the pH. • Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing.

19. The Way stacking gel works.. • When the power is turned on: • Negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. • In this environment glycine switches predominantly to the zwitterionic (neutrally charged) state. • This loss of charge causes them to move very slowly in the electric field.

20. 2. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine. 3. The separation of Cl- from the Tris counter-ion (which is now moving towards the cathode) creates a narrow zone with a steep voltage gradient that pulls the glycine along behind it, resulting in two narrowly separated fronts of migrating ions; • the highly mobile Cl- front, followed by the slower, mostly neutral glycine front.

21. 4. All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl- • so when the two fronts sweep through the sample well the proteins are concentrated into the narrow zone between the Cl- and glycine fronts.

22. 5. This procession carries on until it hits the running gel, • where the pH switches to 8.8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. • So the glycine front accelerates past the proteins, leaving them behind. 6. The result is that the proteins are dumped in a very narrow band at the interface of the stacking and running gels. • Since the running gel has an increased acrylamide concentration, which slows the movement of the proteins according to their size, the separation begins…

23. What if not using a stacking gel? • In the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep. • Rather than being lined up together and hitting the running gel together, the proteins in the sample would all enter the running gel at different times, resulting in very smeared bands. • So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.

24. The Running Gel.. • Separation • Once the proteins are in the running gel, they are separated because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins. • The size of the pores in the gel can be altered depending on the size of the proteins you want to separate by changing the acrylamide concentration.

26. Kaleidoscope Standard

27. Polyacrylamide gel polymerisation • Polyacrylamide, used mainly for SDS-PAGE, • Is a matrix formed from monomers of acrylamide and bis-acrylamide. • It’s strengths are that is it chemically inert – so won’t interact with proteins as they pass through – and that it can easily and reproducibly be made with different pore sizes to produce gels with different separation properties.

28. The polymerisation reaction, shown in the diagram below, is a vinyl addition catalysed by free radicals. • The reaction is initiated by TEMED, which induces free radical formation from ammonium persulphate (APS). • The free radicals transfer electrons to the acrylamide/bisacrylamide monomers, radicalizing them and causing them to react with each other to form the polyacrylamide chain.

29. In the absence of bis-acrylamide, the acrylamide would polymerise into long strands, not a porous gel. • But as the diagram shows, bis-acrylamide cross-links the acrylamide chains and this is what gives rise to the formation of the porous gel matrix. • The amount of crosslinking, and therefore the pore size and consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bis-acrylamide.